Donor-strand exchange drives assembly of the TasA scaffold in Bacillus subtilis biofilms

Many bacteria in nature exist in multicellular communities termed biofilms, where cells are embedded in an extracellular matrix that provides rigidity to the biofilm and protects cells from chemical and mechanical stresses. In the Gram-positive model bacterium Bacillus subtilis, TasA is the major protein component of the biofilm matrix, where it has been reported to form functional amyloid fibres contributing to biofilm structure and stability. Here, we present electron cryomicroscopy structures of TasA fibres, which show that, rather than forming amyloid fibrils, TasA monomers assemble into fibres through donor-strand exchange, with each subunit donating a β-strand to complete the fold of the next subunit along the fibre. Combining electron cryotomography, atomic force microscopy, and mutational studies, we show how TasA fibres congregate in three dimensions to form abundant fibre bundles that are essential for B. subtilis biofilm formation. Our study explains the previously observed biochemical properties of TasA and shows how a bacterial extracellular globular protein can assemble from monomers into β-sheet-rich fibres, and how such fibres assemble into bundles in biofilms.

Line 277-format of eps needs amending Lines 379,396,446 and Table S3 (perhaps other places as well)-Typo: 361O should be 3610 if this is referring to NCIB 3610 Methods: Add in % w/v or v/v as needed in the methods. Figure 5. Missing scale bars for pellicles and AFM images. Are the AFM images also done using height mode? Seems like there should be a height scale for each as is done in Figure S7. Why was the 'WT' strain used for AFM a TasA-mCherry strain? There is also no reference to the creation of this strain. Was it produced for this study? Is TasA still functional? Is the fusion stable and how does the mCherry integrate into the structure of the fibre? Figure 6. Although it is only an illustration, panel C is somewhat inaccurate and thus loses usefulness. The cells are not proportional to the fibres as evident from the EM images in the paper. The cells are spaced much differently than seen in the EM images as well. Perhaps a model that looks more akin to the data would be a suitable replacement. Moreover, do you have a perspective on if the fibres are anchored to the cell body or mainly released based on your imaging? It was unclear if the cells that were not covered or touching the TasA fibres were supposed to not be producers and not coated or if the TasA fibres are secreted fully. Table S2. Should add the reference for the paper in which each strain was created. Figure S2 contains a panel about a possible divalent metal cation but this is not mentioned in the text at all.  Comment about data accessibility-The accessibility of the raw data and datasets in the paper is currently challenging. The models of protein structures and images should be submitted to repositories. It is also not clear which strains the authors are referring to at time. Please link to the strain table more explicitly in figure legends and methods etc.
In many places there is no reference to how many biological and technical replicates have been completed. Examples: pellicles, colony biofilms, AFM samples… Where these only completed once or are they representative images of a dataset? Nicola Stanley-Wall and Natalie Bamford Reviewer #2 (Remarks to the Author): The manuscript by Böhning et al. describes the molecular architecture of TasA, a fibre forming protein produced by the soil bacterium Bacillus subtilis. Importantly, TasA is a major protein component of the extracellular matrix in B. subtilis biofilms, and it contributes to the structure and rigidity of these multicellular assemblies. Previous studies reported a high-resolution X-ray structure of monomeric TasA and it has been speculated that TasA forms filaments with characteristics of functional amyloids. Another study, however, suggested that TasA fibres consist of a linear arrangement of globular subunits, without structural rearrangements between the monomeric and filamentous form of TasA.
Böhning et al. applied helical reconstruction from cryoEM images of purified TasA filaments to address this ambiguity. The authors reached a resolution of sub-4Å and were able to show that TasA filaments were hold together by an unexpected N-terminal beta-strand exchange. These subunit interactions are reminiscent to what was observed in other bacterial filaments, such as type I pili, or other eukaryotic filaments, like uromodulin. This is a major new insight, of broad interest and beautifully described!
The authors further set out to analyze the interaction between TasA fibres by using a combination of cryo-electron tomography, atomic force microscopy as well as by the mutation of specific residues. This led to the identification of specific interaction sites involved in filament-bundling and the analysis of the created mutants revealed defects in biofilm formation.
While the results on the structure and assembly of TasA filaments are impressive and of high quality, this referee is not yet fully convinced about the second major part of this manuscript, covering the analysis of the interaction between TasA filaments in vitro and in biofilms. The following major comments should be addressed before publication: Major comments: -This reviewer is not an expert in phase-separation, however, the authors did not manage to convincingly show that TasA fibres spontaneously associate into phaseseparated bundles, as stated in the title of Figure 3 and in line 175-177. What is the difference between protein aggregation into highly-ordered filament bundles and phase separation? What are the main characteristics to call this phase separation? The authors should discuss these concerns in the manuscript. Furthermore, if the authors resist on making this statement, they should include accompanying experiments, such as demonstrating concentration dependence of filament bundling. Does the assembly of bundles from different stock concentrations result in the same phase separation/filament bundling? If I understood correctly, a nematic phase is a meta state between a crystal and an isotropic liquid. Would a fourier transform of a projection image of the crystal-like bundles show discrete spots? -The analysis of the interaction sites between a TasA doublet using atomic model fitting into a noisy tomogram density is flawed and should be removed or supported by a subtomogram average of the filament doublet. If you look closely in Figure 4c, you can even see that the fit of the lower filament, at the filament contact site right of the black box is off. This flawed fitting of the atomic structure is not even needed, as the geometrical considerations shown in Figure 4e nicely demonstrates that the loop with residues 174-177 resides furthest away from the helical centre, indicating potential interaction sites, which are further supported by the MD simulations. On top of that, the interaction between filaments in bundles seem to be different compared to the one in doublets. Whereas in doublets, contact sites only exist in the periphery, filaments nicely align along their long axis in bundles. How can this be explained? Would it be possible to calculate a subtomogram average of bundles to visualize the three-dimensional arrangement and compare it to the interaction of filaments observed in doublets? % <FC CTMNCOOGLK ICRCIO LD <?O1 GK PFC JQP?KPO P?O1 W',*%',, ?KB P?O1 W(-%)-OCCJ PL be significantly reduced compared to wild-type when looking at the SDS page and Western blot in Figure S6b,c. Might this be a reason for the observed phenotypes in biofilm and pellicle formation? This should be discussed in the manuscript. Is it possible that these mutants never reach the required TasA concentration to form bundles? The cryoEM images in Figure S6d are of such poor quality in the provided PDF file, that it is impossible to properly proof the authors conclusion, especially in their example of tasA W(-%)-& 6CNC$ OLJC DGI?JCKPO ?NC L@OCNR?@IC$ @QP GP OCCJO PL @C ?P ? BGDDCNCKP OA?IC0 7 could not find any statement on the length of the scale bars in this panel. % <FC ANUL49 GJ?ECO LD PFC 2& OQ@PGIGO WOGK: WCMO @GLDGIJO GK 5GEQNC +E$F ?KB 5GEQNC ;are of rather poor quality. Why did the authors not apply cryoET for visualization? This could even highlight potential interactions between TasA fibres and B. subtilis cells. Would it be possible to use cryo-focused ion beam milling to thin unperturbed biofilms and to subsequently perform cryoET of biofilms in a more native state?
Minor comments: - Figure 1a: The cryoEM image is very pixelated and it is unclear if the data is of this poor quality or if it is an artifact of PDF compression. A higher resolution image with a larger field of view together with a Zoom in would allow for a proper quality assessment of the raw data.
- Figure S2c: This panel with the putative metal ion is neither cited nor discussed in the text.
- Figure 2a,c: The indication of the N-and C-terminus in the structure would facilitate a quicker understanding of this figure.
- Figure 2b: The schematic is hard to understand, due to the two grey boxes. This could be understood as two TasA subunits, however, in my understanding it should represent one TasA monomer whit a schematic of the donor strand exchange.
- Figure S2d, Figure S4: The authors frequently jump between different panels of Figure  S2 and S3 which makes it hard to follow. Can the panel S2d be included in Figure S4? - Figure 3: Similar to Fig. 1a, the provided images are pixelated, however, in movie S3 the quality of the data seems to be good. The arrow pointing to the fibre bundle in 3b seems to be a bit shifted.
- Figure 4: The arrow pointing to the doublet seems to be off target and a bit shifted.
- Figure S5: Please indicate residue numbers in your model. -Line 213: The statement on the observation of TasA in AFM images should be toned down. e.g.: we observed filament networks potentially formed by TasA fibres adherend PL ACIIO PF?P ?NC KLP MNCOCKP GK WP?O1 @GLDGIJO& - Figure S7: The color of scale bars could be changed to white.
- Figure 5h: The name of the two 2D classes "This sample" and "Donor-strand TasA" might be inaccurate and should be changed to a more descriptive title.
Reviewer #3 (Remarks to the Author): The manuscript "Molecular architecture of the TasA scaffold in Bacillus subtilis biofilms" by Bohning, Ghrayeb, Pedebos, Abbas, Khalid, Chai and Bharat, describes the fibres forming biofilms and built from the assembly of TasA monomers. Cryo-EM puts in evidence a new way of assembling through donor strand complementation.
The manuscript content is very interesting and deserves certainly publication. Concerning the coarse-grained molecular dynamics simulations, I was a bit puzzled by the short lengths of the trajectories. Indeed, looking in the literature, coarse-grained molecular dynamics often represent several microseconds or tenths of microseconds. Maybe, the big size (10 millions atoms) of the considered system prevents the authors to record such long trajectories, but I would strongly suggest that they extend the length of their simulations and also record some few more.
Another remark concerning the trajectories is that they are almost not analyzed. Classical analyses as coordinate RMSD could be included in the SI. It would be interesting to know how large the relative postions of TasA monomers deform during the trajectories. Also, the authors point out interacting residues belonging to different fibres ( Figure S5), but do not tell much how the interactions take place: are they direct interactions, or mediated by ions or water molecules? Are the interactions established between different fibres or within the same fibre? Do they residue involved in interaction play a role in the establishment of biofilms, or have another functional role or are conserved in the sequence?

Reviewer #1 (Remarks to the Author)"
"Molecular architecture of the TasA scaffold in Bacillus subtilis biofilms" Böhning et al. present an atomic model of the biofilm matrix fibre TasA as determined by cryo-EM. They show that the fibres isolated have the same structure as those of biofilm samples using cryo-electron tomography. The fibres were found to be formed by donor-strand exchange like some bacterial pili. These results are important to the field and settle the ongoing controversy over whether these biofilm fibres were amyloid in character or not. The authors present well founded evidence that the TasA fibres, ?IQFLREF Z%OGAF$ ?OC KLK%?JVILECKGA& =C @CIGCSC QF?Q QFGP TLOH GP LD EOC?Q S?IRC QL QFC fields of biofilm and protein fibre research.
Overall, the manuscript is well written with helpful, well-constructed figures. Below we have outlined some possible improvements and suggestions to improve the readability of the manuscript but believe that no further experiments are required for this story.
We thank the reviewers for their helpful comments.

Comments:
Suggest hyphenating "donor-strand" as this is commonly done in literature We have implemented this change as suggested.
Line 50-53. The way this sentence is worded suggests that biofilms are surface attached communities, but the definition is broader than this and includes cell aggregates in solution for instance.
We have adjusted the wording of the sentence to address your valid point: L51: "Biofilms form in environmental settings as well as inside the bodies of living organisms during infections, and are further commonly found on abiotic surfaces such as medical devices." Line 70. No need to write out Bacillus subtilis anymore.
Thank you -we have adjusted this.
Line 88. Suggest adding that the X-ray diffraction studies were on in situ TasA fibres. Otherwise, the paper referenced in the previous line also showed that there was no AOLPP%Z M?QQCOK GK QFC >%O?V BGDDO?AQGLK LD <?P1 DG@OGIP& We have added this.
L88: "Moreover, a recent X-ray diffraction study showed that native TasA fibrils only produce a weak cross-f-sheet pattern in vitro and in situ." 9GKC ..& 8Q JGEFQ @C JLOC ?AARO?QC QL P?V QF?Q QFC DG@OGIP XMOLBRAC ? TC?H AOLPP%Z%PFCCQ X-ray pattern" rather than "possess" We agree that this is more accurate and have implemented this wording.
Line 106-107 Remove statements like "to our surprise" -this is subjective and do not need to be included in the reporting.
Removed as suggested.
We have simplified this description as follows: L147: "In the fibre form, the previously self-complementing f-strand (residues 48-61) is displaced by the donor-strand of the (n-1)th subunit (residues 28-38). The previously self-complementing strand then folds over the opposite f-sheet, and the donor-strand (residues 28-38), along with residues 39-47, extends towards the next (n+1)th subunit, allowing assembly of the next subunit ( Figure 2a)." Line 176. Are you suggesting that all the strands are parallel (no anti-parallel arrangement)? Could you state this more clearly if this is so? This is a very good question. The resolution of our tomograms does not allow us to unambiguously assign polarities. To answer this question, we have attempted subtomogram averaging (STA) of fibres in bundles, which we now include in the revised manuscript as Figure S5b. Unfortunately, STA of fibres in bundles did not allow us to resolve interactions between fibres, likely due to their dynamic and flexible nature. Fourier transforms (power spectra shown) of bundles reveal a wide range of spots (revised Figure S5a), indicating that fibre-fibre interactions are not rigid.
To probe this further, we have performed MD simulations for antiparallel fibres interactions, which also produced a stable solution, presented in the revised Figure  S6, showing how the outer loop residues help in this interaction.
After submission of this manuscript, another study was published by Ed Egelman's lab 1 showing that, in TasA-like fibres from hyperthermophilic archaea (termed 'archaeal bundling pili', ABP), arrange themselves in five parallel strands that interact with one antiparallel strand to form distinct, six-fibre bundles. There is significant structural similarity between TasA and ABP fibres, including a donor-strand exchange. Compared to TasA, however, ABP fibres form ordered, six-fibre bundles. In TasA, however, bundles of various sizes are seen, and the assembly is less ordered. We discuss this within the manuscript now.
To summarise, we think that the interaction of fibres could be either parallel or anti-parallel. TasA filaments from different cells within the biofilm might stack through parallel or anti-parallel interactions, embedding the cells within the matrix. We have now added the above information into the paper: L266: "We were not able to resolve distinct interactions between fibres using STA, which, in addition to lack of distinct lattice spots in Fourier transforms of bundles, suggests that interactions between fibres are not highly ordered. This interaction appears to be qualitatively similar to formation of tactoids by the phage Pf4 in P. aeruginosa biofilms 2 . Nevertheless, all our experiments show the importance of a loop (containing residues 174-177) extending away from the fibre surface in mediating these interactions. Interestingly, a system was recently described in hyperthermophilic archaea (ABP pilus system), with significant structural and sequence homology to TasA 1 . The ABP filaments further formed ordered, six-fibre bundles of similar morphology as TasA. While highly ordered interactions appear to exist within the ABP bundles, we could not detect a fixed size for TasA bundles, which appear to be more flexible. These stronger interactions in ABP filaments may be required as the archaea containing ABP live in acidic and boiling waters. The exact mode of interaction between TasA fibres at the atomic structural level remains to be determined, however our MD simulations suggest that both antiparallel and parallel TasA bundling could be possible." Line 183. Sentence starts with "Our fits into…" and this seems to be missing a word.
'Fit' was meant as a noun here -we, however, realise the potential for confusion. Given a comment by reviewer #2, we have removed this sentence completely.
Does the 174-177 alanine variant suffer from any stability issues? It looks like there is less protein isolated from the biofilm according to the western blot. Can you report # of 174-177AAAA fibres in figure S6 f?
We thank the reviewer for pointing this out, which we realise was not very clear in the original manuscript. The SDS-PAGE and Western blot images were originally added to verify the presence of TasA in our preparations. However, these gels were not quantitative as they showed different amounts of protein from different purifications, where concentrations were not normalised. To account for the expression levels of TasA in the wild-type and mutant strains, we now show an SDS-PAGE gel with protein preparations after the first purification step from the same number of cells (revised Figure S7b). To compare the fibre properties in vitro by wild-type and mutant proteins, we used the same initial concentrations. Here, TasA appears to be present in approximately equivalent amounts.
Interestingly, despite the similar concentrations used, we did detect lower numbers of fibres formed in purifications in the 174-177AAAA mutant compared to the wild-type ( Figure S7c), and those fibres bundled at a lower percentage compared to wild-type. This information is now also plotted in Figure S7d. It may well be that the 174-177AAAA mutant fibres are slightly less stable -we discuss this now: L201: "Also, cryo-EM of the 174-177AAAA mutant fibres showed markedly reduced bundling ( Figure S7c-d, 24% wild-type versus 2% for the mutant); however, this correlated with a smaller amount of TasA fibres formed by the mutant, as indicated by less fibres being detectable in the sample overall, despite the use of the same concentration of TasA." however at the level of strains being able to cross complementing each other there are several other papers. We suggest you remove this concluding sentence or you will need to ground your conclusion more firmly in the previous literature.
Thank you -we have removed this sentence as suggested in the main text and cited the two papers in the discussion. We have fixed this -our apologies.
Methods: Add in % w/v or v/v as needed in the methods.
We have added this. Figure 5. Missing scale bars for pellicles and AFM images. Are the AFM images also done using height mode? Seems like there should be a height scale for each as is done in Figure S7. Why was the 'WT' strain used for AFM a TasA-mCherry strain? There is also no reference to the creation of this strain. Was it produced for this study? Is TasA still functional? Is the fusion stable and how does the mCherry integrate into the structure of the fibre?
We have added scale bars in the pellicle images. Regarding AFM images: AFM images can be taken using different modes. The most conventional mode is height mode but, when the purpose of the images is to show qualitatively the presence of certain features, it is much more informative to use 'peak force error' mode. Images taken in the latter mode do not have a real height scale and therefore it is not presented in Figure 5. To provide information about the height scale, we added into the revised Figure S8, the same AFM images that are shown in Figure  5, that were taken simultaneously in height mode, and hence they include a height scale. Please note that details observed in the fibres taken in peak force error mode are not as apparent in these height images and this is why we chose 'peak force error' mode images for the main text and figures. We have added an explanation to the choice of AFM modes in the experimental section-L509: "Peak force error images do not provide information on height and therefore they are presented without a height scale. However, with each peak force error image we also present the corresponding height sensor image in the extended data." Regarding the use of a TasA-mCherry strain: we employ a fluorescent strain as it allows us to quickly confirm via fluorescence microscopy whether TasA is appropriately expressed. The used strain was constructed for a different study 3 and the ZK strain collection number (ZK5041) was specified in Table S3. We have now added a reference as well. We further add supporting evidence -showing the fluorescence microscopy images of cells within the biofilm (Figure S8g-i) and the biofilm itself compared to a wild-type ( Figure S8j-k) -showing that the wildtype phenotype is retained in the TasA-mCherry strain. Figure 6. Although it is only an illustration, panel C is somewhat inaccurate and thus loses usefulness. The cells are not proportional to the fibres as evident from the EM images in the paper. The cells are spaced much differently than seen in the EM images as well. Perhaps a model that looks more akin to the data would be a suitable replacement. Moreover, do you have a perspective on if the fibres are anchored to the cell body or mainly released based on your imaging? It was unclear if the cells that were not covered or touching the TasA fibres were supposed to not be producers and not coated or if the TasA fibres are secreted fully.
We have updated the figure to produce a more accurate representation of the AFM data, with denser cells and networks of TasA bundles at more accurate scale. We unfortunately have no structural data on how fibres are anchored to the cell yet -would be an important subject for future inquiries. Previous literature suggests that TasA fibres emanate from cells 4 , but we cannot comment on how cells and bundles interact with our data in this manuscript. Table S2. Should add the reference for the paper in which each strain was created. Table S2 outlines the primers used to design new strains that were used in this study. References were indeed missing in Table S3 and they have now been added. Figure S2 contains a panel about a possible divalent metal cation but this is not mentioned in the text at all.
We now mention this in the text: L78: "Interestingly, a density in the cryo-EM map that may represent a cation, coordinated by two negatively charged aspartate (Asp) residues, was observed ( Figure S2c), which may be an ancestral remnant of the camelysin family of metalloproteinases, from which TasA has been suggested to derive 5 ." Figure S4 -Would be helpful to add residue numbers for the sequence alignment.
We agree and have implemented this. Figure S5 -Can you indicate which loop is which, perhaps with lines/arrows and number ranges above them?
We have added these into the revised Figure S6 as requested. Figure S6. There is no length given for the scale bars in panel D.
Our apologies -this has been added to the caption in the revised Figure S7.

Comment about data accessibility-
The accessibility of the raw data and datasets in the paper is currently challenging. The models of protein structures and images should be submitted to repositories.
We fully agree -we have now deposited the atomic model and map to the PDB and EMDB respectively, with the accession numbers 8AUR and EMD-15673. We further attach the atomic model and cryo-EM map with this submission.
It is also not clear which strains the authors are referring to at time. Please link to the strain table more explicitly in figure legends and methods etc.
We have now specifically noted the strain number each time it is mentioned in the methods section and we refer to the strain list and strain numbers in the figure legends.
In many places there is no reference to how many biological and technical replicates have been completed. Examples: pellicles, colony biofilms, AFM samples… Where these only completed once or are they representative images of a dataset?
All the pellicle, biofilm and AFM images represent a dataset. We have now added the number of biological repeats and technical replicates to the methods section.

Reviewer #2 (Remarks to the Author):
The manuscript by Böhning et al. describes the molecular architecture of TasA, a fibre forming protein produced by the soil bacterium Bacillus subtilis. Importantly, TasA is a major protein component of the extracellular matrix in B. subtilis biofilms, and it contributes to the structure and rigidity of these multicellular assemblies. Previous studies reported a high-resolution X-ray structure of monomeric TasA and it has been speculated that TasA forms filaments with characteristics of functional amyloids. Another study, however, suggested that TasA fibres consist of a linear arrangement of globular subunits, without structural rearrangements between the monomeric and filamentous form of TasA.
Böhning et al. applied helical reconstruction from cryoEM images of purified TasA filaments to address this ambiguity. The authors reached a resolution of sub-4Å and were able to show that TasA filaments were hold together by an unexpected Nterminal beta-strand exchange. These subunit interactions are reminiscent to what was observed in other bacterial filaments, such as type I pili, or othser eukaryotic filaments, like uromodulin. This is a major new insight, of broad interest and beautifully described!
The authors further set out to analyze the interaction between TasA fibres by using a combination of cryo-electron tomography, atomic force microscopy as well as by the mutation of specific residues. This led to the identification of specific interaction sites involved in filament-bundling and the analysis of the created mutants revealed defects in biofilm formation.
While the results on the structure and assembly of TasA filaments are impressive and of high quality, this referee is not yet fully convinced about the second major part of this manuscript, covering the analysis of the interaction between TasA filaments in vitro and in biofilms. The following major comments should be addressed before publication: We thank the reviewer for their helpful comments. We have incorporated the suggestions and toned down the messaging in the second half of the manuscript, clarifying what we can and cannot say given the data at hand. We also provide some additional experimental data to support our claims.
Major comments: -This reviewer is not an expert in phase-separation, however, the authors did not manage to convincingly show that TasA fibres spontaneously associate into phaseseparated bundles, as stated in the title of Figure 3 and in line 175-177. What is the difference between protein aggregation into highly-ordered filament bundles and phase separation? What are the main characteristics to call this phase separation? The authors should discuss these concerns in the manuscript. Furthermore, if the authors resist on making this statement, they should include accompanying experiments, such as demonstrating concentration dependence of filament bundling. Does the assembly of bundles from different stock concentrations result in the same phase separation/filament bundling? If I understood correctly, a nematic phase is a meta state between a crystal and an isotropic liquid. Would a fourier transform of a projection image of the crystal-like bundles show discrete spots?
We thank the reviewer for this comment, and fully understand the reviewer's request to clarify. We termed the observed bundling of TasA fibres 'phase separation' in the original manuscript since it is highly reminiscent of the formation of liquid crystals (a form of phase separation) by the filamentous Pf4 phage, a structural component in P. aeruginosa biofilms 2 . The term 'liquid crystal' would imply that the individual fibres are orientationally aligned, but positionally random, which is the definition of a nematic liquid crystalline phase, i.e., no discrete spots corresponding to a crystalline lattice seen in a Fourier transform.
We have projected tomograms of TasA bundles and performed Fourier transforms, and indeed, we rather see a wide range of indistinct signal, but no discrete spots corresponding to a higher order lattice, apart from those corresponding to the helical repeat of TasA fibres (revised Figure S5). We believe this also has implications for how rigid fibre-fibre interactions in bundles are, which we discuss in response to the reviewer's next comment.
We do agree, though, that to prove that this is an example of liquid-liquid phase separation will require detailed experiments. Since this is not really the focus of the manuscript, we will remove this statement. In the revised manuscript, we have removed any mention of 'phase separation' and merely refer to the similarities with other systems in the discussion.
L269: "This interaction appears to be qualitatively similar to formation of tactoids by the phage Pf4 in P. aeruginosa biofilms 2 ." -The analysis of the interaction sites between a TasA doublet using atomic model fitting into a noisy tomogram density is flawed and should be removed or supported by a subtomogram average of the filament doublet. If you look closely in Figure 4c, you can even see that the fit of the lower filament, at the filament contact site right of the black box is off. This flawed fitting of the atomic structure is not even needed, as the geometrical considerations shown in Figure 4e nicely demonstrates that the loop with residues 174-177 resides furthest away from the helical centre, indicating potential interaction sites, which are further supported by the MD simulations. On top of that, the interaction between filaments in bundles seem to be different compared to the one in doublets. Whereas in doublets, contact sites only exist in the periphery, filaments nicely align along their long axis in bundles. How can this be explained? Would it be possible to calculate a subtomogram average of bundles to visualize the three-dimensional arrangement and compare it to the interaction of filaments observed in doublets? This is an important point, and we agree that the fit into the bundles is not as clear as our atomic structural data. As requested, we have performed subtomogram averaging (STA) of TasA fibres exclusively picked in the centre of bundles. Despite our best attempts, our STA refinements only resolve a single TasA fibre in the centre of the bundle, with no ordered density of fibres around them (revised Figure S5). We also tried to subclassify the data, but did not obtain any interacting fibres around the central fibre. This is consistent with the Fourier transform of the bundle (power spectra shown; Figure S5) showing no distinct repeats corresponding to an even partially crystalline lattice, and suggesting a more dynamic interaction.
In accordance with the reviewer's suggestion, we only show an overlay of our atomic TasA model with a cryo-ET slice, and refer to our data showing the loop is the most extended element from the fibre centre in the main text. Given we were not able to resolve an STA of fibres in bundles, we believe that the periodic interaction observed in doublets, which was also seen in MD, is not rigid, as one would expect in a crystal lattice; we hope that further studies can shed light on the exact nature of this interaction.
It is interesting to note that, after we submitted this manuscript and uploaded it to bioRxiv, another manuscript was published by Ed Egelman's lab showing the structure of TasA-like bundling pili in extremophilic archaea 1 (please see also our response to reviewer 1 above). The study shows that these so-called ABP pili are structurally similar to TasA, consisting of donor-strand exchanged subunits, and interact periodically within bundles, similar to what we observe for TasA fibre doublets. However, in contrast to TasA, ABP bundles distinctly consist of six fibres, and the structure of such six-pili bundles could be resolved to near-atomic resolution using helical reconstruction. We cannot resolve such fibre interactions for TasA, and neither is the size of the bundles fixed, suggesting variable or weaker interactions. As the reviewer stated, the fact that the loop containing residues 174-177 extends the farthest from the helical axis, that the residues interact in our MD simulations, and that mutating these residues has a profound effect of biofilm formation, suggests that these are key for forming filament-filament interfaces. We have updated the discussion in light of the above.
L266: "We were not able to resolve distinct interactions between fibres using STA, which, in addition to lack of distinct lattice spots in Fourier transforms of bundles, suggests that interactions between fibres are not highly ordered. This interaction appears to be qualitatively similar to formation of tactoids by the phage Pf4 in P. aeruginosa biofilms 2 . Nevertheless, all our experiments show the importance of a loop (containing residues 174-177) extending away from the fibre surface in mediating these interactions. Interestingly, a system was recently described in hyperthermophilic archaea (ABP pilus system), with significant structural and sequence homology to TasA 1 . The ABP filaments further formed ordered, six-fibre bundles of similar morphology as TasA. While highly ordered interactions appear to exist within the ABP bundles, we could not detect a fixed size for TasA bundles, which appear to be more flexible." The reviewer also mentions that it appears in tomograms of TasA bundles that the filaments are aligned along their long axis rather than interacting periodically. We think this could just be a visual effect: Fibres also appear to be aligned in micrographs of ABP ( Figure 1 of Wang et al.), but the ABP atomic-resolution structure reveals that fibre-fibre interactions within the bundle are purely periodic (Figure 3 of Wang et al.).
% <FC CUMOCPPGLK ICSCIP LD <?P1 GK QFC JRQ?KQP Q?P1 Y'-*%'--?KB Q?P1 Y(.%). PCCJ to be significantly reduced compared to wild-type when looking at the SDS page and Western blot in Figure S6b,c. Might this be a reason for the observed phenotypes in biofilm and pellicle formation? This should be discussed in the manuscript. Is it possible that these mutants never reach the required TasA concentration to form bundles?
We thank the reviewer for pointing this out, which we realise was not very clear in the original manuscript. The SDS-PAGE and Western blot images were originally added to verify the presence of TasA in our preparations. However, these gels were not quantitative as they showed different amounts of protein from different purifications where concentrations were not normalised. To account for the expression levels of TasA in the wild-type and mutant strains, we now show an SDS-PAGE gel with protein preparations after the first purification step from the same number of cells (revised Figure S7b). Here, TasA appears to be present in approximately equivalent amounts.
To compare the fibre properties in vitro by wild-type and mutant proteins, we used the same initial concentrations. Interestingly, we did detect lower numbers of fibres in purifications also in the 174-177AAAA mutant compared to the wild-type ( Figure  S7c), and those fibres bundled at a lower percentage compared to wild-type. This information is now also plotted in Figure S7d. It may well be that the 174-177AAAA mutant fibres are slightly less stable -we discuss this now: L201: "Also, cryo-EM of the 174-177AAAA mutant fibres showed markedly reduced bundling ( Figure S7c-d, 24% wild-type versus 2% for the mutant); however, this correlated with a smaller amount of TasA fibres formed by the mutant, as indicated by less fibres being detectable in the sample overall, despite the use of the same concentration of TasA." The cryoEM images in Figure S6d are of such poor quality in the provided PDF file, that it is impossible to properly proof the authors conclusion, especially in their CU?JMIC LD Q?P1 Y(.%).& 7COC$ PLJC DGI?JCKQP ?OC L@PCOS?@IC$ @RQ GQ PCCJP QL @C ?Q a different scale? I could not find any statement on the length of the scale bars in this panel.
Apologies -the annotation for the scale bar has been added in the figure caption. We have now filtered the images differently to make the fibres more readily visible, but the fibres have a fairly small diameter (~4 nm) and it thus hard to achieve high contrast. We thank the reviewer for this suggestion. Some of the filamentous structures present GK WtasA may be flagella, which are abundant in B. subtilis biofilms and can also be PCCK GK QFC AOVL%4: GJ?EGKE B?Q? LD Weps WsinR biofilms in Figure S9. As for the lack of vesicles etc., in the AFM images -sample preparation for AFM imaging includes several washing steps that would most likely eliminate all the elements that are not adsorbed on the mica surface. We are unsure whether the features observed are really vesicles or some other molecules in a different orientation. 1P OCNRCPQCB$ TC F?SC MCODLOJCB 15: LD Weps WsinR pellicles. Significantly increased KRJ@COP LD DGI?JCKQLRP PQORAQROCP A?K @C PCCK ALJM?OCB QL WtasA (revised Figure  S8). We added a paragraph to the manuscript: L212: "Next, by imaging intact wild-type pellicle biofilms using atomic force microscopy (AFM), we observed networks of filaments adherent to cells, potentially formed by <?P1 DG@OCP$ QF?Q ?OC PCSCOCIV OCBRACB GK WtasA biofilms (Figure 5e-f; Figure S8). Similarly, greatly increased numbers of fibres could again be seen in a strain that overproduces TasA but lacks eps "WsinR Weps) ( Figure S8e-f)." % <FC AOVL4: GJ?ECP LD QFC 2& PR@QGIGP YPGK; YCMP @GLDGIJP GK 5GEROC +E$F ?KB 5GEROC S8 are of rather poor/quality. Why did the authors not apply cryoET for visualization? This could even highlight potential interactions between TasA fibres and B. subtilis cells. Would it be possible to use cryo-focused ion beam milling to thin unperturbed biofilms and to subsequently perform cryoET of biofilms in a more native state?
We have replaced potentially low-resolution images -we hope these will be an improvement. We did acquire some cryo-ET data but its appearance is overall worse than of the shown high-dose 2D images. Particularly, the B. subtilis cells are considerably too thick to reconstruct good tomograms without prior thinning.
We think the possibility of performing cryo-FIB milling is an excellent point and would likely be required to resolve interactions between the cell and the fibre bundles. We are looking to address this in future studies, since this would be a significant undertaking beyond the scope of this paper on the TasA atomic structure.
Minor comments: - Figure 1a: The cryoEM image is very pixelated and it is unclear if the data is of this poor quality or if it is an artifact of PDF compression. A higher resolution image with a larger field of view together with a Zoom in would allow for a proper quality assessment of the raw data.
We have now replaced the image with a differently filtered image and hope that it appears improved to the reviewer -we think that this image is representative of the raw cryo-EM data, which was acquired relatively close to focus (between -1 and -2 µm defocus). As with cryo-EM data of small fibres (the diameter of a TasA subunit is less than 4 nm), the raw micrographs are intrinsically noisy, but we hope that the image conveys the wave-like shape of the TasA fibre.
- Figure S2c: This panel with the putative metal ion is neither cited nor discussed in the text.
We now mention it within the Results section: L125: "Interestingly, a density in the cryo-EM map that may represent a cation, coordinated by two negatively charged aspartate (Asp) residues, was observed ( Figure S2c), which may be an ancestral remnant of the camelysin family of metalloproteinases, from which TasA has been suggested to derive 5  Thank you, we agree that this makes the structural re-arrangement more easily visible. We have implemented this into the figure.
- Figure 2b: The schematic is hard to understand, due to the two grey boxes. This could be understood as two TasA subunits, however, in my understanding it should represent one TasA monomer whit a schematic of the donor strand exchange.
Indeed, the grey boxes were meant to represent the individual beta-sheets. We have now adjusted this to indicate the subunit more clearly with an additional box and adjusted the description within the figure.
- Figure S2d, Figure S4: The authors frequently jump between different panels of Figure S2 and S3 which makes it hard to follow. Can the panel S2d be included in Figure S4?
We have added panel S2d to Figure S4.
- Figure 3: Similar to Fig. 1a, the provided images are pixelated, however, in movie S3 the quality of the data seems to be good.
We have replaced these with differently filtered images -the quality should be comparable to the movie now.
The arrow pointing to the fibre bundle in 3b seems to be a bit shifted.
Thanks -this is now adjusted.
- Figure 4: The arrow pointing to the doublet seems to be off target and a bit shifted.
We have adjusted this.
- Figure S5: Please indicate residue numbers in your model.
We have added the numbers into the revised Figure S6 as requested.
-Line 207 and Figure S6d: It seems that the authors collected cryoEM images of TasA DG@OCP "TQ ?KB JRQ?KQP# TFGAF T?P MROGDGCB DOLJ 2& PR@QGIGP YPGK; YCMP JRQ?KQP& <FGP should be clearly stated in the manuscript as well as in the figure panel and not only in the legend and methods part.
We now mention this in the main text and in the legend for Figure 1. L101: "To gain insights into the structure of TasA fibres, we purified TasA from B. subtilis W#&' ('$%" using previously established procedures 6 ." -Line 213: The statement on the observation of TasA in AFM images should be toned down. e.g.: we observed filament networks potentially formed by TasA fibres adherend QL ACIIP QF?Q ?OC KLQ MOCPCKQ GK YQ?P1 @GLDGIJP& We have adjusted this statement as suggested. L212: "Next, by imaging intact wild-type pellicle biofilms using atomic force microscopy (AFM), we observed networks of filaments adherent to cells, potentially formed by <?P1 DG@OCP$ QF?Q ?OC PCSCOCIV OCBRACB GK WtasA biofilms (Figure 5e-f; Figure S8). Similarly, greatly increased numbers of fibres could again be seen in a strain that overproduces TasA but lacks eps "WsinR Weps) ( Figure S8e-f)." - Figure S7: The color of scale bars could be changed to white.
We have updated this (now Figure S8).
- Figure 5h: The name of the two 2D classes "This sample" and "Donor-strand TasA" might be inaccurate and should be changed to a more descriptive title.
We have updated this and now use the terms 'biofilm sample' and 'in vitro sample'.

Reviewer #3 (Remarks to the Author):
The manuscript "Molecular architecture of the TasA scaffold in Bacillus subtilis biofilms" by Bohning,Ghrayeb, Pedebos, Abbas, Khalid, Chai and Bharat, describes the fibres forming biofilms and built from the assembly of TasA monomers. Cryo-EM puts in evidence a new way of assembling through donor strand complementation.
The manuscript content is very interesting and deserves certainly publication.
We thank the reviewer for their comments.
Concerning the coarse-grained molecular dynamics simulations, I was a bit puzzled by the short lengths of the trajectories. Indeed, looking in the literature, coarse-grained molecular dynamics often represent several microseconds or tenths of microseconds. Maybe, the big size (10 millions atoms) of the considered system prevents the authors to record such long trajectories, but I would strongly suggest that they extend the length of their simulations and also record some few more.
We have now extended the simulations to 500 ns and recorded triplicates -these are now shown in the revised Figure S6. The results and inferences from the simulations are the same as reported in the first version of the manuscript.
Another remark concerning the trajectories is that they are almost not analyzed. Classical analyses as coordinate RMSD could be included in the SI. It would be interesting to know how large the relative postions of TasA monomers deform during the trajectories. Also, the authors point out interacting residues belonging to different fibres ( Figure S5), but do not tell much how the interactions take place: are they direct interactions, or mediated by ions or water molecules? Are the interactions established between different fibres or within the same fibre? Do they residue involved in interaction play a role in the establishment of biofilms, or have another functional role or are conserved in the sequence?
We have now annotated the image interactions further and added RMSD plots as well as incorporated RMSF data in Figure S6. The interactions are between residues of different fibres and direct protein-protein interactions (information now added to the figure caption). We believe that the outermost loop residues, which play an important role in fibre-fibre interaction are key to the establishment of biofilms, because mutating them prevents efficient biofilm formation (see Figures 5a and S7a)